Molecular Breeding

, Volume 23, Issue 2, pp 289–298 | Cite as

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

  • Huijun Zhang
  • Hezhong Dong
  • Weijiang Li
  • Yi Sun
  • Shouyi Chen
  • Xiangqiang Kong


Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T3 generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l−1 NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.


Glycine betaine Choline monooxygenase Salt stress AhCMO transgenic cotton 



The research was supported financially by a Project for cotton (nyhyzx07-005-02) from Chinese Ministry of Agriculture. We also gratefully acknowledge the financial support from Agricultural Seed Industrialization Foundation of Shandong Province (2005-cotton), and the High Innovation Fund (2006YCX009) from Shandong Academy of Agricultural Sciences. We thank Prof. A. E. Eneji of China Agricultural University for critical reading of the manuscript.


  1. Ahmad S, Khan N, Iqbal MZ, Hussain A, Hassan M (2002) Salt tolerance of cotton (Gossypium hirsutum L.). Asian J Plant Sci 1:715–719Google Scholar
  2. Arakawa K, Katayama M, Takabe T (1990) Levels of betaine and betaine aldehyde dehydrogenase activity in the green leaves and etiolated leaves and roots of barley. Plant Cell Physiol 31:797–803Google Scholar
  3. Ashraf M (2002) Salt tolerance of cotton: some new advances. Crit Rev Plant Sci 21:1–30. doi: 10.1016/S0735-2689(02)80036-3 CrossRefGoogle Scholar
  4. Blunden G, Patel AV, Armstrong NJ, Gorham J (2001) Betaine distribution in the Malvaceae. Phytochemistry 58:451–454. doi: 10.1016/S0031-9422(01)00263-1 PubMedCrossRefGoogle Scholar
  5. Bohnert HJ, Shen B (1999) Transformation and compatible solutes. Sci Hortic (Amsterdam) 78:237–260. doi: 10.1016/S0304-4238(98)00195-2 CrossRefGoogle Scholar
  6. Cha-um S, Supaibulwatana K, Kirdmanee C (2006) Water relation, photosynthetic ability and growth of Thai Jasmine rice (Oryza sativa L. ssp. Indica Cv. KDML 105) to salt stress by application of exogenous glycinebetaine and choline. J Agron Crop Sci 192:25–36. doi: 10.1111/j.1439-037X.2006.00186.x CrossRefGoogle Scholar
  7. Chen SY, Zhu LH, Hong J (1991) Molecular biology identification of a salt-tolerant rice line. Acta Bot Sin 33:569–573Google Scholar
  8. Chomczynski P, Sacci N (1987) Single-step method of RNA isolation by acid guanidiumthiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi: 10.1016/0003-2697(87)90021-2 PubMedCrossRefGoogle Scholar
  9. Flower TG, Yeo AR (1986) Ion relations of plant drought and salinity. Aust J Plant Physiol 13:75–91CrossRefGoogle Scholar
  10. Gibon Y, Bessieres MA, Larher F (1997) Is glycine betaine a non-compatible solute in higher plants that do not accumulate it? Plant Cell Environ 20:329–340. doi: 10.1046/j.1365-3040.1997.d01-82.x CrossRefGoogle Scholar
  11. Gorham J (1996) Glycine betaine is a major nitrogen-containing solute in the Malvaceae. Phytochemistry 43:367–369. doi: 10.1016/0031-9422(96)00312-3 CrossRefGoogle Scholar
  12. Gossett DR, Millhollon EP, Lucas MC (1994a) Antioxidant response to NaCl stress in salt-tolerant and saltsensitive cultivars of cotton. Crop Sci 34:706–714Google Scholar
  13. Gossett DR, Millhollon EP, Lucas MC, Banks SW, Marney MM (1994b) The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cultivars (Gossypium hirsutum L.). Plant Cell Rep 13:498–503. doi: 10.1007/BF00232944 CrossRefGoogle Scholar
  14. Grumet R, Hanson AD (1986) Glycine-betaine accumulation in barley. Aust J Plant Physiol 13:353–364Google Scholar
  15. Guo BH, Zhang YM, Li HJ, Du LQ (2000) Transformation of wheat with a gene encoding for the betaine aldehyde dehydrogenase (BADH). Acta Bot Sin 42:279–283Google Scholar
  16. Guo Y, Zhang L, Xiao G, Chen SY (1997) Expression of the BADH gene and salinity tolerance in rice transgenic plants. Sci China 27:151–155Google Scholar
  17. Iturbe-Ormaetxe I, Escuredo PR, Arrese-Igor C, Becana M (1998) Oxydative damage in pea plants exposed to water deficit or paraquat. Plant Physiol 116:173–181. doi: 10.1104/pp.116.1.173 CrossRefGoogle Scholar
  18. Johnson GN, Young AJ, Scholes JD, Horton P (1993) The dissipation of excess excitation energy in British plant species. Plant Cell Environ 16:673–679. doi: 10.1111/j.1365-3040.1993.tb00485.x CrossRefGoogle Scholar
  19. Lv S, Yang A, Zhang K, Wang L, Zhang J (2007) Increase of glycinebetaine synthesis improves drought tolerance in cotton. Mol Breed 20:233–248. doi: 10.1007/s11032-007-9086-x CrossRefGoogle Scholar
  20. Matoh T, Watanabe J, Takahashi E (1987) Sodium, potassium, chloride, and betaine concentrations in isolated vacuoles from salt-grown Atriplex gmelini leaves. Plant Physiol 84:173–177PubMedCrossRefGoogle Scholar
  21. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668. doi: 10.1093/jexbot/51.345.659 PubMedCrossRefGoogle Scholar
  22. McCue KF, Hanson AD (1992) Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression. Plant Mol Biol 18:1–11. doi: 10.1007/BF00018451 PubMedCrossRefGoogle Scholar
  23. Meek CR, Oosterhuis D (2000) Effects of glycine betaine and water regime on diverse cotton cultivars. Proceedings of the 2000 cotton Research Meeting. AAES Special Report 198: 109–112Google Scholar
  24. Meloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ Exp Bot 49:69–76. doi: 10.1016/S0098-8472(02)00058-8 CrossRefGoogle Scholar
  25. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant cDNA. Nucleic Acids Res 8:4321–4325. doi: 10.1093/nar/8.19.4321 PubMedCrossRefGoogle Scholar
  26. Naidu BP, Cameron DF, Konduri SV (1998) Improving drought tolerance of cotton by glycine betaine application and selection. In: Proceedings of the 9th Australian agronomy conference, Wagga Wagga.
  27. Premachandra GS, Ogata S, Saneoka H (1989) Evaluation of polyethylene glycol test for measurement of cell membrane stability in maize. Soil Sci Plant Nutr 35:565–573Google Scholar
  28. Quan RD, Shang M, Zhang H, Zhao YX, Zhang JR (2004) Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnol J 2:477–486. doi: 10.1111/j.1467-7652.2004.00093.x PubMedCrossRefGoogle Scholar
  29. Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol 44:357–384. doi: 10.1146/annurev.pp.44.060193.002041 CrossRefGoogle Scholar
  30. Rhodes D, Rich PJ, Brunk DG, Ju GC, Rhodes JC, Pauly MH, Hansen LA (1989) Development of two isogenic sweet corn hybrids differing for glycine betaine content. Plant Physiol 91:1112–1121PubMedCrossRefGoogle Scholar
  31. Russell BL, Rathinasabapathi B, Hanson AD (1998) Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranch. Plant Physiol 116:859–865. doi: 10.1104/pp.116.2.859 PubMedCrossRefGoogle Scholar
  32. Sairam RK, Tyagi A (2004) Physiology and molecular biology of salinity stress tolerance in plants. Curr Sci 86:407–421Google Scholar
  33. Sakamoto A, Murata N (2001) The use of bacterial choline oxidase, a glycine betaine synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol 125:180–188. doi: 10.1104/pp.125.1.180 PubMedCrossRefGoogle Scholar
  34. Sakamoto A, Murata N (2002) The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ 25:163–171. doi: 10.1046/j.0016-8025.2001.00790.x PubMedCrossRefGoogle Scholar
  35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. America Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  36. Shen YG, Du BX, Zhang WK, Zhang JS, Chen SY (2002) AhCMO, regulated by stress in Atriplex hortensis, can improve drought tolerance in transgenic tobacco. Theor Appl Genet 105:815–821. doi: 10.1007/s00122-002-1006-1 PubMedCrossRefGoogle Scholar
  37. Sulpice R, Tsukaya H, Nonaka H, Mustardy L, Chen TH, Murata N (2003) Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine. Plant J 36:165–176. doi: 10.1046/j.1365-313X.2003.01873.x PubMedCrossRefGoogle Scholar
  38. Tang QY, Feng MG (1997) Practical statistics and DPS data processing system. China Agricultural Press, Beijing, pp 1–407Google Scholar
  39. Tang W, Luo Z, Wen SM, Dong HZ, Xin CS, Li WJ (2007) Comparison of inhibitory effects on leaf photosynthesis in cotton seedlings between drought and salinity stress. Cotton Sci 19:28–32Google Scholar
  40. Xiao G, Zhang GY, Liu FH, Chen SY (1995) The study of the BADH gene in Atriplex hortensis. Chin Sci Bull 40:741–745Google Scholar
  41. Xing W, Rajashekar CB (2000) Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana. Environ Exp Bot 46:21–28. doi: 10.1016/S0098-8472(01)00078-8 CrossRefGoogle Scholar
  42. Yancey PH (1994) Compatible and counteracting solutes. In: Strange K (ed) Cellular and molecular physiology of cell volume regulation. CRC Press, Boca Raton, pp 81–109Google Scholar
  43. Zhang HJ, Dong HZ, Shi YJ, Chen SY, Zhu YH (2007) Transformation of cotton (Gossypium hirsutum) with AhCMO gene and the expression of salinity tolerance. Acta Agron Sin 33:1073–1078Google Scholar
  44. Zhu JK (2001) Over expression of a delta-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water and salt stress in transgenic rice. Trends Plant Sci 6:66–72. doi: 10.1016/S1360-1385(00)01838-0 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Huijun Zhang
    • 2
  • Hezhong Dong
    • 1
  • Weijiang Li
    • 1
  • Yi Sun
    • 3
  • Shouyi Chen
    • 4
  • Xiangqiang Kong
    • 1
  1. 1.Cotton Research Center, Shandong Key Lab for Cotton Culture and PhysiologyShandong Academy of Agricultural SciencesJinanChina
  2. 2.Cotton Research InstituteShanxi Academy of Agricultural SciencesYunchengChina
  3. 3.The Agriculture Biotechnology Research Center of Shanxi ProvinceTaiyuanChina
  4. 4.Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingChina

Personalised recommendations